1. Technical Field
The present invention relates, in general, to the molecular cloning and expression of a receptor protein, and, in particular, to a prostaglandin F2xcex1 receptor and fragments thereof linked to the activation of second messengers as measured, for example, by cAMP, IP3 or intracellular calcium. The invention further relates to a DNA sequence encoding a prostaglandin F2xcex1 receptor, to a recombinant DNA molecule that includes such a DNA sequence and to cells transformed therewith. The invention also relates to antibodies directed against the F2xcex1 receptor and to a method of detecting an F2xcex1 receptor with the antibody. The invention further relates to a method of detecting the presence of an F2xcex1 receptor encoding a DNA fragment in a sample, the use of transformed cells for screening drugs, as well as to drugs prepared using such a screening method.
2. Background Information
Prostaglandin F2xcex1 receptors belong to a large class of hormone receptors which are linked to their signal transduction pathways via guanine nucleotide binding regulatory (G) proteins. Such receptors are amongst the most intensively studied receptor systems. Prostaglandin receptors have been classically defined as being linked to the stimulation of second messengers and measured by cyclic AMP (cAMP), inositol 3-phosphate (IP3) or intracellular calcium and are coupled with a G regulatory protein (Muallem, Biochem. J. 263: 769-774 (1989)). In contrast, activation of prostaglandin receptors may result in various responses, including inhibition of adenylyl cyclase activity, inhibition of phosphatidylinositol turnover and inhibition of Ca2+ mobilization (Muallem, Biochem. J. 263: 769-774 (1989), and Duncan, Endocrinology 128: 1519-1526 (1991)). Evidence has also accumulated suggesting heterogeneity in the category of receptors (Balapure, Biol. Reprod. 41: 385-392 (1989)).
Two prostaglandin receptors have previously been cloned, viz. the human and mouse thromboxane A2 receptor and the mouse prostaglandin E3 receptor (Hirata, Nature 349: 617-620 (1991); Namba, BBRC 184: 1197-1203 (1992); and Sugimoto, J. Biol. Chem. 267: 6463-6466 (1992), respectively).
Prostaglandin F2xcex1 receptors are extremely important from a clinical therapeutic viewpoint. Drugs which activate (agonists) these receptors may be used to treat glaucoma (Alm, Arch. Ophthalmol. 109:1564-1568 (1991)), whereas drugs which block (antagonists) prostaglandin F2xcex1 receptors may be used therapeutically to treat pathological conditions, e.g. in the lungs and uterus. It may be of pharmaceutical value to be able to titer endogenous prostaglandin F2xcex1 with a solubilized receptor as well as to use an immobilized receptor in the purification of a ligand and its analogs. Despite their clinical utility, one problem with the prostaglandin F2xcex1 agonist and putatively antagonist drugs currently available, is that they have many side effects, like many other drugs which work through interaction with receptors. These side effects are predominantly due to a lack of receptor specificity. That is, the drug in use interacts not only with prostaglandin F2xcex1 receptors but with other receptors as well, see e.g. Muallem, Biochem. J. 263;769-774 (1989).
A major goal of clinical pharmacology and the pharmaceutical industry is the development of more selective drugs with greater efficacy than those currently in use. Impediments to this process are the low abundance of prostaglandin F2xcex1 receptor protein available to study in eye tissue and the lack of suitable homogeneous model systems of the receptors with which to screen drugs against.
The present invention seeks to provide a solution to this problem by a novel approach which comprises cloning cDNAs encoding prostaglandin F2xcex1 receptors, constructing eukaryotic expression vectors containing these cDNAs, and creating a series of stably transfected mammalian cell lines or prokaryotic cells which express functional prostaglandin F2xcex1 receptors in high abundance. These cell lines, which would express a homogeneous population of prostaglandin F2xcex1 receptors, can be used by the pharmaceutical industry or others to screen drugs and study the prostaglandin F2xcex1 receptors using a variety of biochemical, physiological and pharmacological techniques.
To accomplish this goal, we have isolated a cDNA encoding a rat prostaglandin F2xcex1 receptor subtype linked to the activation of second messengers as measured by e.g. cAMP, IP3 or intracellular calcium. This cDNA encoding an F2xcex1 receptor is inserted into different eukaryotic and prokaryotic expression vectors and used in the construction of various mammalian cell lines expressing this functional protein. Resulting F2xcex1 receptor-expressing cell lines can be used to investigate the affinities and efficacies of agonist and antagonist drugs with an F2xcex1 receptor using various techniques, such as radioligand binding and second messenger assays.
One aspect of the present invention therefore relates to an F2xcex1 receptor that is linked to the stimulation of second messengers, such as cAMP, IP3 or intracellular calcium, and that couples with guanine nucleotide binding regulatory (G) proteins, when present.
Another aspect of the present invention relates to a DNA fragment encoding the above described prostaglandin F2xcex1 receptor.
A further aspect of the present invention relates to a recombinant DNA construct or molecule comprising a vector and the above-described DNA fragment.
Yet another aspect of the present invention relates to a host cell transformed with the above described recombinant DNA construct.
In another aspect, the present invention relates to a process of producing the above-described prostaglandin F2xcex1 receptor. The method comprises culturing the above-mentioned host cell under conditions such that the F2xcex1 receptor encoding DNA fragment is expressed and a prostaglandin F2xcex1 receptor is produced.
Still another aspect of the invention relates to an antibody directed against the F2xcex1 receptor.
Another aspect of the invention relates to a method of detecting the presence of an F2xcex1 receptor in a sample by contacting the sample with such an antibody.
Still another aspect of the invention relates to a method of detecting the presence in a sample of a DNA fragment encoding an F2xcex1 receptor by contacting the sample with a DNA probe comprising a DNA fragment encoding an F2xcex1 receptor protein or polypeptide to hybridize the DNA fragment thereto.
Yet another aspect of the invention relates to a method of screening drugs for F2xcex1 receptor activating or blocking activity by contacting the above-mentioned transformed host cell with the drugs.
Another aspect of the invention relates to a method of preparing a drug, which method includes screening drug candidates for F2xcex1 receptor activating or blocking activity.
A further aspect of the invention relates to a drug, the preparation of which included screening drug candidates for F2xcex1 receptor activating or blocking activity.